Previous work has demonstrated that bovine brain myosin II heavy chains migrate as a doublet in SDS-PAGE (Murakami et al., FEBS Lett. 278: 23, 1991). We analyzed purified bovine brain myosin using antibodies raised to the carboxyl-terminal amino acid sequence of nonmuscle myosin heavy chain-B as well as to a sequence of 21 amino acids that is inserted in the head region of myosin heavy chain-B near the actin binding site (Takahashi et al., J. Biol. Chem. 267: 17864, 1992). Whereas the former antibody was capable of detecting both myosin heavy chain-B isoforms, the antibody to the insert only detected the slower migrating myosin heavy chain-B. These results are similar to results which were obtained in this laboratory using extracts of human brains except that, in this case, it was also possible to recognize a myosin heavy chain-A isoform. In vitro phosphorylation of bovine brain myosin using protein kinase C showed that both myosin heavy chains could be phosphorylated. Analysis of the myosin heavy chain isoforms by phosphopeptide mapping showed two different patterns. A single tryptic phosphopeptide was generated from the faster migrating myosin heavy chain-B isoform and this phosphopeptide comigrated with a hexapeptide originating from the carboxyl-terminus of a myosin heavy chain with a known amino acid sequence. Based on the comigration of the two peptides, both in isoelectric focusing as well as following two-dimensional peptide mapping, we predict the sequence of this peptide to be -glu-val-ser-thr-leu-lys- wherein either the serine or the threonine is phosphorylated. The slower migrating myosin heavy chain-B appears to be phosphorylated within the 21 amino acid insert present in the amino-terminal head region. Moreover, the generation of brain heavy meromyosin, which lacks the carboxyl-terminal PKC phosphorylation site, results in a myosin heavy chain that still can undergo phosphorylation. Digestion of the phosphorylated myosin heavy chain by trypsin resulted in a single major myosin heavy chain phosphopeptide. This peptide comigrates with a phosphopeptide obtained by proteolysis of a synthetic peptide duplicating the inserted sequence that had been previously phosphorylated by PKC. Present studies are directed towards understanding what the effect of phosphorylation of protein kinase C is in vitro and are also directed at obtaining evidence as to whether and under what circumstances this phosphorylation may occur in intact cells.